A Natural Vibrio parahaemolyticus Δ pirA ^Vp pirB ^Vp+ Mutant Kills Shrimp but Produces neither Pir ^Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions

Abstract

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirAVp and PirBVp. These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirAVp but carried pirBVp. Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirBVp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirAVp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirAVp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirAVp gene sequence and in the downstream pirBVp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirABVp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no PirVp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirAVp and PirBVp. The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND. Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND. Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.

pirA‐ and pirB‐like gene identification in Micrococcus luteus strains in Mexico

“…On the other hand, the failure to detect the pirA-like gene with the AP3 primers may be due to what the designers of the AP4 primers postulate, that is, that they obtained false-negative results with AP3 when the samples were preserved at freezing temperatures (Dangtip et al, 2015), as was the case with our samples. Also, similar to that reported for Vibrio parahaemolyticus by Phiwsaiya et al (2017), the PirA-like gene present in the Micrococcus luteus strains could be altered, so the primers do not match correctly.…”

Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus

“…V. parahaemolyticus is a marine bacterial pathogen that can not only cause acute damage to the hepatopancreas organ of shrimp [44][45][46] but also cause gastroenteritis in humans [47][48][49]. Moreover, many Vibrio species are highly resistant to many commercially available antibiotics [50][51][52][53].…”

Characterization of calcineurin A and B genes in the abalone, Haliotis diversicolor, and their immune response role during bacterial infection

“…To investigate the expression patterns of HcCNA and HcCNB in response to bacterial challenge, thirty-six abalones were randomly divided into two groups; the bacterial challenge and saline control groups. V. parahaemolyticus, XN89, previously isolated from diseased shrimp (Phiwsaiya et al, 2017) was recovered from our −80 • C frozen storage by streaking onto tryptic soy agar (TSA) supplemented with 1.5% NaCl followed by incubation at 30 • C overnight. Bacterial culture was then prepared by inoculating a single colony in 5 ml of tryptic soy broth (TSB) with 1.5% NaCl for 4 h at 30 • C with shaking.…”